The objectives of this research are: (1) to define the diversity among antibodies specific for the group A streptococcal cell wall carbohydrate (A CHO); (2) to assess genetic mechanisms responsible for generating the diversity among these antibodies; (3) to evaluate the roles of ontogeny, antigenic stimulation and isotype switching in the diversification process; and (4) to relate this information to idiotypic and antigen-binding properties of A CHO-specific antibodies. In the past year, we have determined that only two germ line structural genes encode the light chain variable regions of 90% of the anti-A CHO antibodies produced by strain A mice. We have, likewise, determined that two to five genes encode the heavy chain variable regions of these antibodies. In the coming year, we will focus on isolating these genes using conventional DNA cloning procedures. Comparison of the nucleotide sequences of these genes isolated from A CHO antibody-producing hybridomas with that of the corresponding genes isolated from germ line DNA will reveal the respective roles of somatic mutation and combinatorial joining in producing diversity. We plan to continue developing A CHO hybridomas representative of primary, secondary and hyperimmune antibody responses for the purpose of examining the roles of antigenic stimulation and isotype switching on the diversification of antibodies. In addition, we are attempting to obtain A CHO hybridomas from young mice to assess the influence of ontogeny on repertoire expression and diversification.